Method for treatment of neurodegenerative diseases

ABSTRACT

Patients suffering from neurodegenerative diseases are treated by administering an effective amount of a gamma-nicotine compound.

BACKGROUND OF THE INVENTION

The present invention relates to a method for treating patients havingneurodegenerative diseases, and in particular, to a method for treatingpatients suffering from those diseases which cause a cholinergicdeficit.

Senile dementia of the Alzheimer's type (SDAT) is a debilitatingneurodegenerative disease, mainly afflicting the elderly; characterizedby a progressive intellectual and personality decline, as well as a lossof memory, perception, reasoning, orientation and judgment. One featureof the disease is an observed decline in the function of cholinergicsystems, and specifically, a severe depletion of cholinergic neurons(i.e., neurons, that release acetylcholine, which is believed to be aneurotransmitter involved in learning and memory mechanisms). See,Jones, et al., Intern. J. Neurosci., Vol. 50, p. 147 (1990); Perry, Br.Med. Bull., Vol. 42, p. 63 (1986) and Sitaram, et al., Science, Vol.201, p. 274 (1978). It has been observed that nicotinic acetylcholinereceptors, which bind nicotine and other nicotinic agonists with highaffinity, are depleted during the progression of SDAT. See, Giacobini,J. Neurosci. Res., Vol. 27, p. 548 (1990); and Baron, Neurology, Vol.36, p. 1490 (1986). As such, it would seem desirable to providetherapeutic compounds which either directly activate nicotinic receptorsin place of acetylcholine or act to minimize the loss of those nicotinicreceptors.

Parkinson's disease (PD) is a debilitating neurodegenerative disease,presently of unknown etiology, characterized by tremors and muscularrigidity. A feature of the disease appears to involve the degenerationof dopaminergic neurons (i.e., which secrete dopamine). One symptom ofthe disease has been observed to be a concomitant loss of nicotinicreceptors which are believed to modulate the process of dopaminesecretion. See, Rinne, et al., Brain Res., Vol. 54, pp. 167-170 (1991)and Clark, et al., Br. J. Pharm., Vol. 85, pp. 827-835 (1985).

Certain attempts have been made to treat SDAT. For example, nicotine hasbeen suggested to possess an ability to activate nicotinic cholinergicreceptors upon acute administration, and to elicit an increase in thenumber of such receptors upon chronic administration to animals. See,Rowell, Adv. Behav. Biol., Vol. 31, p. 191 (1987); and Marks, J.Pharmacol. Exp. Ther., Vol. 226, p. 817 (1983). Other studies indicatethat nicotine can act directly to elicit the release of acetylcholine inbrain tissue, to improve cognitive functions, and to enhance attention.See, Rowell, et al., J. Neurochem., Vol. 43, p. 1593 (1984); Hodges, etal., Bio. of Nic., Edit. by Lippiello, et al., p. 157 (1991); Sahakian,et al., Br. J. Psych., Vol. 154, p. 797 (1989); and U.S. Pat. No.4,965,074 to Leeson.

It would be desirable to provide a method for treating neurodegenerativediseases, such as SDAT and PD, by administering a nicotinic compound tothe patient suffering from such disease.

SUMMARY OF THE INVENTION

The present invention relates to a method for the treatment of aneurodegenerative disease. The method involves treating a patientsuffering from such disease (e.g., SDAT or PD) with an effective amountof a gamma-nicotine compound.

The method of the present invention provides benefits to the patient inthat the compounds have the potential to (i) act as a pharmacologicalagonist to activate nicotinic receptors, and (ii) elicitneurotransmitter secretion. In addition, the compounds are expected tohave the potential to (i) increase the number of nicotinic cholinergicreceptors of the brain of the patient, and (ii) exhibit neuroprotectiveeffects.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a method for the treatment ofneurodegenerative diseases, such as SDAT and PD. In particular, themethod involves treating a patient with an effective amount of acompound having the general formula: ##STR1## where R represents H oralkyl, such as straight chain or branched alkyl (e.g., C₁ to about C₅,or other lower alkyl). Preferably, R is methyl. One or more of thecarbon atoms of the pyridine ring and/or one or more of the carbon atomsof the pyrolidine ring can include substituent groups other thanhydrogen (e.g., alkyl or halo, such as F, Cl, Br or I, substituents inthe case of the pyridine ring; and alkyl substituents in the case of thepyrolidine ring). Representative alkyl substituents are previouslydefined. The compounds can be employed as racemic mixtures or asenanitomers. See Registry Nos. 5860-66-2 and 71606-36-5; Seeman, et al.,J. Org. Chem., Vol 46, p. 3040 (1981) and Glenn, et al., J. Org. Chem.,Vol. 43, p. 2860 (1978).

The manner in which the compounds are administered can vary. Thecompounds can be administered by inhalation; in the form of an aerosoleither nasally or using delivery articles of the type set forth in U.S.Pat. No. 4,922,901 to Brooks, et al. and U.S. patent application Ser.No. 486,025, filed Feb. 27, 1990; orally (e.g., in liquid form within asolvent such as an aqueous liquid, or within a solid carrier);intravenously (e.g., within a saline solution); or transdermally (e.g.,using a transdermal patch). Exemplary methods for administering suchcompounds will be apparent to the skilled artisan. Certain methodssuitable for administering compounds useful according to the presentinvention are set forth in U.S. Pat. No. 4,965,074 to Leeson. Theadministration can be intermittent, or at a gradual, continuous,constant or controlled rate to a warm-blooded animal, such as a humanbeing or other mammal.

The dose of the compound is that amount effective to treat theneurodegenerative disease from which the patient suffers. By "effectiveamount" or "effective dose" is meant that amount sufficient to passacross the blood-brain barrier of the patient, to bind to relevantreceptor sites in the brain of the patient, and to elicitneuropharmacological effects (e.g., elicit neurotransmitter secretion,thus resulting in effective treatment of the disease). Treatment of aneurodegenerative disease involves a decrease of symptoms of theparticular disease.

For human patients, the effective dose of typical compounds generallydoes not exceed about 150 μg, often does not exceed about 100 μg, andfrequently does not exceed about 50 μg, per kg patient weight. For humanpatients, the effective dose of typical compounds generally is at leastabout 5 μg, often is at least about 10 μg, and frequently is at leastabout 25 μg, per kg of patient weight For human patients, the effectivedose of typical compounds generally requires administering the compoundin an amount of at least about 2 0, often at least about 1.0, andfrequently at least about 0.1 mg/hr./patient. For human patients, theeffective dose of typical compounds requires administering the compoundin an amount which generally does not exceed about 10, often does notexceed about 5, and frequently does not exceed about 2.5 mg/hr./patient.

The compounds useful according to the method of the present inventionhave the ability to pass across the blood-brain barrier of the patientAs such, such compounds have the ability to enter the central nervoussystem of the patient. The log P values of typical compounds useful incarrying out the present invention generally are greater than 0, oftenare greater than about 0.1, and frequently are greater than about 0.5.The log P values of such typical compounds generally are less than about3.0, often are less than about 2.5, and frequently are less than about2.0. Log P values provide a measure of the ability of a compound to passacross a diffusion barrier, such as a biological membrane. See, Hansch,et al., J. Med. Chem., Vol. 11, p. 1 (1968).

The compounds useful according to the method of the present inventionhave the ability to bind to, and hence cause activation of, nicotiniccholinergic receptors of the brain of the patient. As such, suchcompounds have the ability to act as nicotinic agonists. The receptorbinding constants of typical compounds useful in carrying out thepresent invention generally exceed about 1 nM, often exceed about 200nM, and frequently exceed about 500 nM. The receptor binding constantsof such typical compounds generally are less than about 10 μM, often areless than about 7 μM, and frequently are less than about 2 μM. Receptorbinding constants provide a measure of the ability of the compound tobind to half of the relevant receptor sites of certain brain cells ofthe patient. See, Cheng, et al., Biochem. Pharmacol., Vol. 22, pp.3099-3108 (1973).

The compounds useful according to the method of the present inventionhave the ability to demonstrate a nicotinic function by effectivelyeliciting neurotransmitter secretion from nerve ending preparations(i.e., synaptosomes). As such, such compounds have the ability to causerelevant neurons to release or secrete acetylcholine, dopamine, andother neurotransmitters. Generally, typical compounds useful in carryingout the present invention provide for the secretion of dopamine inamounts of at least about 5 percent, often at least about 25 percent,and frequently at least about 50 percent, of that elicited by an equalmolar amount of S(-) nicotine.

The following example is provided in order to further illustrate variousembodiments of the invention but should not be construed as limiting thescope thereof. Unless otherwise noted, all parts and percentages are byweight.

EXAMPLE 1

Mice (DBA strain) were maintained on a 12 hour light/dark cycle and wereallowed free access to water and food supplied by Wayne Lab Blox,Madison, Wis. Animals used in the present studies were 60 to 90 days ofage and weighed 20 to 25 g. Brain membrane preparations were obtainedfrom pooled brain tissue of both males and females.

Mice were killed by cervical dislocation. Brains were removed and placedon an ice-cold platform. The cerebellum was removed and the remainingtissue was placed in 10 volumes (weight:volume) of ice-cold buffer(Krebs-Ringers HEPES:NaCl, 118 mM; KCl, 4.8 mM; CaCl₂, 2.5 mM; MgSO₄,1.2 mM; HEPES, 20 mM; pH to 7.5 with NaOH) and homogenized with aglass-Teflon tissue grinder. The resulting homogenate was centrifuged at18000×g for 20 min. and the resulting pellet was resuspended in 20volumes of water. After 60 min. incubation at 4° C., a new pellet wascollected by centrifugation at 18000×g for 20 min. After resuspension in10 volumes of buffer, a new final pellet was again collected bycentrifugation at 18000×g for 20 min. Prior to each centrifugation step,the suspension was incubated at 37° C. for 5 min. to promote hydrolysisof endogenous acetylcholine. The final pellet was overlayered withbuffer and stored at - 70° C. On the day of the assay, that pellet wasthawed, resuspended in buffer and centrifuged at 18000×g for 20 min. Theresulting pellet obtained was resuspended in buffer to a finalconcentration of approximately 5 mg protein/ml. Protein was determinedby the method of Lowry, et al., J. Biol. Chem., Vol. 193, pp. 265-275(1951), using bovine serum albumin as the standard.

The binding of L-[³ H]nicotine was measured using a modification of themethod of Romano, et al., Science, Vol. 210, pp. 647-650 (1980) asdescribed previously by Marks, et al., Mol. Pharmacol., Vol. 30, pp.427-436 (1986). The binding of L-[³ H]nicotine was measured using a 2hr. incubation at 4° C. Incubations contained about 500 μg of proteinand were conducted in 12 mm×75 mm polypropylene test tubes in a finalincubation volume of 250 μl. The incubation buffer was Krebs-RingersHEPES containing 200 mM TRIS buffer, pH 7.5. The binding reaction wasterminated by filtration of the protein containing bound ligand ontoglass fiber filters (Micro Filtration Systems) that had been soaked inbuffer containing 0.5 percent polyethyleneimine. Filtration vacuum was-50 to -100 torr. Each filter was washed five times with 3 ml ofice-cold buffer The filtration apparatus was cooled to 2° C. before useand was kept cold through the filtration process. Nonspecific bindingwas determined by inclusion of 10 μM nonradioactive nicotine in theincubations. The inhibition of L-[³ H]nicotine binding by test compoundswas determined by including one of eight different concentrations of thetest compound in the incubation. Inhibition profiles were measured using10 nM L-[³ H]nicotine and IC₅₀ values were estimated as theconcentration of compound that inhibited 50 percent of specific L-[³H]nicotine binding. Inhibition constants (Ki values) were calculatedfrom the IC₅₀ values using the method of Cheng, et al., Biochem.Pharmacol., Vol. 22, pp. 3099-3108 (1973). The Ki values for allcompounds for which an inhibition constant less than 100 μM wasdetermined from the inhibition curves described above were alsocalculated independently using Dixon plots for inhibition measured using2 nM, 8 nM and 20 nM concentrations of L-[³ H]nicotine. The L-[³H]nicotine used in all experiments was purified chromatographically bythe method of Romm, et al., Life Sci., Vol, 46, pp. 935-943 (1990).

Log P values (log octanol/water partition coefficient), which have beenused to assess the relative abilities of compounds to pass across theblood-brain barrier, were calculated according to the methods describedby Hansch, et al., J. Med. Chem., Vol. 11, p. 1 (1968).

Dopamine release was measured by preparing synaptosomes from thestriatal area of rat brain obtained from Sprague-Dawley rats generallyaccording to the procedures set forth by Nagy, et al., J. Neurochem.,Vol. 43, pp. 1114-1123 (1984). Striata from 4 rats were homogenized in 2ml of 0.32M sucrose buffered with 5 mM HEPES (pH 7.5), using aglass-teflon tissue grinder. The homogenate was diluted to 5 ml withadditional homogenization solution and centrifuged at 1000×g for 10 min.This procedure was repeated on the new pellet and the resultingsupernatant was centrifuged at 12,000×g for 20 min. A 3 layerdiscontinuous Percoll gradient consisting of 16 percent, 10 percent and7.5 percent Percoll in HEPES-buffered sucrose was made with the finalpellet dispersed in the top layer. After centrifugation at 15,000×g for20 min., the synaptosomes were recovered above the 16 percent layer witha pasteur pipette, diluted with 8 ml of perfusion buffer (128 mM NaCl,2.4 mM KCl, 3.2 mM CaCl₂, 1.2 mM KH₂ PO₄, 1.2 mM MgSO₄, 25 mM HEPES pH7.4, 10 mM dextrose, 1 mM ascorbate, 0.01 mM pargyline), and centrifugedat 15,000×g for 20 min. The new pellet was collected and re-suspended inperfusion buffer. The synaptosome suspension was incubated for 10 min.at 37° C. Then [³ H]-dopamine (Amersham, 40-60 Ci/mmol) was added to thesuspension to give a final concentration of 0.1 μM in suspension, andthe suspension was incubated for another 5 min. Using this method, 30 to90 percent of the dopamine was taken up into the synaptosomes, asdetermined by scintillation counting following filtration through glassfiber filters soaked with 0.5 percent polyethyleneimine. A continuousperfusion system was used to monitor release following exposure to eachligand (i.e., gamma-nicotine). Synaptosomes were loaded onto glass fiberfilters (Gelman type A/E). Perfusion buffer was dripped onto the filters(0.2-0.3 ml/min.) and pulled through the filters with a peristalticpump. Synaptosomes were washed with perfusion buffer for a minimum of 20min. before addition of the ligand. After the addition of a 0.2 ml of a20 μM solution of ligand, the perfusate was collected into scintillationvials at 1 min. intervals and the dopamine released was quantified byscintillation counting. Peaks of radioactivity released above backgroundwere summed and the average basal release during that time wassubtracted from the total. Release was expressed as a percentage ofrelease obtained with an equal concentration of S(-) nicotine.

Data regarding octanol-water partition coefficients, binding constantsand neurotransmitter secretion capability for the ligands evaluated areset forth in Table I.

                  TABLE I                                                         ______________________________________                                        Compound.sup.1                                                                           Ki (nM).sup.2                                                                            logP    Dopamine Release.sup.3                          ______________________________________                                        gamma-nicotine                                                                           1100 ± 100                                                                            1.3     45                                              ______________________________________                                         .sup.1 Racemic mixture of ligand.                                             .sup.2 Concentration of compound which inhibits 50 percent of L[.sup.3        H]nicotine binding.                                                           .sup.3 Percent release relative to S(-) nicotine.                        

The data in Table I indicate that the compound has the capability ofpassing the blood-brain barrier, binding to high affinity nicotinicreceptors, and eliciting neurotransmitter secretion. Thus, the dataindicate that such compounds have the capability of being useful intreating neurodegenerative diseases.

What is claimed is:
 1. A method for treating a patient suffering fromsenile dementia of the Alzheimer's type, the method comprisingadministering to the patient an effective amount of a compound havingthe formula: ##STR2## where R represents H or alkyl.
 2. The method ofclaim 1 whereby the effective amount of the compound is at least about 5μg/kg patient weight, but does not exceed about 150 μg/kg patientweight.
 3. The method of claim 1 whereby the compound is gamma-nicotinecompound
 4. The method of claim 1 whereby the effective amount ofcompound is administered to the patient in an amount of at least about0.10 mg/hr./patient, but in an amount which does not exceed about 10mg/hr./patient.
 5. A method for treating a patient suffering fromParkinson's disease, the method comprising administering to the patientan effective amount of a compound having the formula: ##STR3## where Rrepresents H or alkyl.
 6. The method of claim 5 whereby the effectiveamount of the compound is at least about 5 μg/kg patient weight, butdoes not exceed about 150 μg/kg patient weight.
 7. The method of claim 5whereby the compound is gamma-nicotine.
 8. The method of claim 5 wherebythe effective amount of compound is administered to the patient in anamount of at least about 0.10 mg/hr./patient, but in an amount whichdoes not exceed about 10 mg/hr./patient.
 9. A method for treating apatient suffering from neurodegenerative disease, the method comprisingadministering to the patient an effective amount of a compound havingthe formula: ##STR4## where R represents H or alkyl.
 10. The method ofclaim 9 whereby the effective amount of the compound is at least about 5μg/kg patient weight, but does not exceed about 150 μg/kg patientweight.
 11. The method of claim 9 whereby the compound isgamma-nicotine.
 12. The method of claim 9 whereby the effective amountof compound is administered to the patient in an amount of at leastabout 0.10 mg/hr./patient, but in an amount which does not exceed about10 mg/hr./patient
 13. The method of claim 1 whereby R represents H or C₁to C₅ alkyl.
 14. The method of claim 5 whereby R represents H or C₁ toC₅ alkyl.
 15. The method of claim 9 whereby R represents H or C₁ to C₅alkyl.
 16. The method of claim 1 whereby the compound includes apyridine ring and a pyrolidine ring; and the pyridine ring includes atleast one halo or C₁ to C₅ alkyl substituent.
 17. The method of claim 5whereby the compound includes a pyridine ring and a pyrolidine ring; andthe pyridine ring includes at least one halo or C₁ to C₅ alkylsubstituent.
 18. The method of claim 9 whereby the compound includes apyridine ring and a pyrolidine ring; and the pyridine ring includes atleast one halo or C₁ to C₅ alkyl substituent.
 19. The method of claim 1whereby the compound includes a pyridine ring and a pyrolidine ring; andthe pyrolidine ring includes at least one halo or C₁ to C₅ alkylsubstituent.
 20. The method of claim 5 whereby the compound includes apyridine ring and a pyrolidine ring; and the pyrolidine ring includes atleast one halo or C₁ to C₅ alkyl substituent.
 21. The method of claim 9whereby the compound includes a pyridine ring and a pyrolidine ring; andthe pyrolidine ring includes at least one halo or C₁ to C₅ alkylsubstituent.
 22. The method of claim 1 whereby the compound includes apyridine ring and a pyrolidine ring; and the pyridine ring includes atleast one halo or C₁ to C₅ alkyl substituent; and the pyrolidine ringincludes at least one C₁ to C₅ alkyl substituent.
 23. The method ofclaim 5 whereby the compound includes a pyridine ring and a pyrolidinering; and the pyridine ring includes at least one halo or C₁ to C₅ alkylsubstituent; and the pyrolidine ring includes at least one C₁ to C₅alkyl substituent.
 24. The method of claim 9 whereby the compoundincludes a pyridine ring and a pyrolidine ring; and the pyridine ringincludes at least one halo or C₁ to C₅ alkyl substituent; and thepyrolidine ring includes at least one C₁ to C₅ alkyl substituent.